Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins

The role of adenoviral capsid protein VI in cell cycle modulation

Les Adénovirus humains sont des virus non enveloppés se répliquant dans le noyau des cellules hôtes.Durant l’infection et après leur entrée par endocytose, les Adénovirus sont transportés au noyau pourinitier l’expression du génome viral. Dans l’endosome, les capsides virales subissent un désassemblagepartiel et libèrent le facteur viral lytique, la protéine VI (pVI). Au niveau de la membrane de l’endosome,cette protéine va alors induire sa rupture permettant ainsi le relargage des virions au sein du cytoplasmegrâce à son hélice amphipatique N-terminale. Par la suite, pVI est transportée vers des structuresnucléaires appelées PML nuclear bodies (PML-NB), associée une ubiquitine ligase cytoplasmique, laNedd4.2. Les PML-NB sont des complexes nucléaires multi-protéiques qui ont des propriétésantivirales. Celles-ci impliquent le recrutement de facteurs de transcription répressifs comme parexemple la protéine anti apoptotique Daxx ou encore le suppresseur de tumeur p53, impliqué dans larégulation du cycle cellulaire. Il a été montré que la protéine pVI en complexe avec Nedd4.2 induit larelocalisation de Daxx des PML-NB dans le cytoplasme, ce qui permet une expression efficace dugénome viral. Ainsi, l’inhibition fonctionnelle de Daxx par pVI suggère que cette protéine virale puisseaussi être impliquée dans la restriction de p53.Dans cette étude, nous avons montré que le nombre des modifications post-traductionnelles (PTM) dep53 augmentent en présence de pVI dans la cellule. De plus, les données obtenues montrent quel’expression de pVI affecte la transcription dépendante de p53 et que l’interaction avec Nedd4.2 n’estpas nécessaire pour inhiber les fonctions de p53. Pour étudier l’implication de pVI dans la modulationdu cycle cellulaire, nous avons créé une lignée cellulaire humaine exprimant cette protéine virale defaçon stable. La caractérisation de cette lignée a permis de mettre en évidence une prolifération cellulaireaccrue. Nos observations ont aussi montré une perte importante des PML-NB et une réduction desprotéines clés du cycle cellulaire p53 et pRb, un autre suppresseur de tumeur. Par des techniques demicro-injection et l’utilisation de l’inhibiteur MG132, nous avons observé que ces deux facteurscellulaires sont ciblés vers le protéasome et dégradés lors de la surexpression de pVI. L’étude desfonctions de cette protéine virale laisse penser que la protéine pVI présente un potentiel oncogéniquecar en effet, sa surexpression induit la dérégulation de l’homéostasie cellulaire et l’inhibition desuppresseurs de tumeur, comme p53 et pRb.Human Adenovirus are non-enveloped viruses which replicate inside the host cell nucleus. Uponinfection and after receptor-mediated entry, they are transported towards the nucleus to initiate the viralgene expression. Viral capsids deliver from the endosome into the cytoplasm by partial disassembly andrelease inside the endosome mediated by viral lytic factor protein VI (pVI). pVI is targeted to themembrane via an amphipathic helix structure in the N-terminus of the viral protein. After membranerupture and capsid release, pVI is transported to sub-nuclear structures, so-called PML nuclear bodies(PML-NBs), together with the cytoplasmic ubiquitin ligase Nedd4.2. PML-NBs represent multiproteinaggregates in the host-cell nucleus with an antiviral capacity, as to several PML-associated repressivetranscription factors, such as the anti-apoptotic Daxx protein and the tumor suppressor p53 were reportedto localize at these foci. In addition, pVI-mediated displacement of Daxx from PML-NBs was shown tooccur in dependency of Nedd4.2 to support efficient viral gene expression. Therefore, we postulate thatbesides Daxx functional inhibition, pVI might also be involved in p53 restriction.Here, we show that p53 posttranslational modification (PTM) is increased when pVI protein is presentin the host-cell. Moreover, we obtained data that pVI expression severely impacts p53 inducedtransactivation of cellular transcription. Biochemical approaches indicate that pVI binding of theubiquitin ligase Nedd4.2 is no prerequisite for the capacity to inhibit p53 functions. In a next step toelucidate the role of pVI on cell cycle regulation, we generated a human cell line stably expressing theviral pVI protein. Our characterization analyses show significantly that these cells benefit from thepresence of pVI as we proved increased cell proliferation rates. We also observed an intense loss ofPML-NBs and reduced protein concentrations of cycle key regulators p53 and pRb. Usingmicroinjection and the inhibitor MG132 we were able to show that both cellular restriction factors weresequestered into the proteasomal degradation pathway of the cell. Evaluation of pVI functions temptedus to speculate, whether pVI might execute oncogenic potential upon overexpression, due toderegulation of host-cell homeostasis and inhibition of tumor suppressive determinants

On the Use of the Sigma-Lognormal Model to Study Children Handwriting

Best paper for innovative researchInternational audienceThis paper investigates the interest of using the Kinematic Theory of rapid human movements to analyseand evaluate the handwriting produced by young kindergarten children in typical classroom environment. A total of66 children participated in this trial. For the preliminary results reported herein, movements from 15 children, takenevenly from three different levels (3, 4, and 5 years old), were analyzed. Our results confirm that 1) the sigmalognormalequation can model accurately childen’s movement and 2) that this modeling can differentiate betweenchildren of different school levels, hence supporting the relevance of pursuing along this original pathway

Critical repolarization gradients determine the induction of reentry-based torsades de pointes arrhythmia in models of long QT syndrome

BACKGROUND Torsades de pointes arrhythmia is a potentially lethal polymorphic ventricular tachyarrhythmia (pVT) in the setting of long QT syndrome. Arrhythmia susceptibility is influenced by risk factors modifying repolarization. OBJECTIVE The purpose of this article was to characterize repolarization duration and heterogeneity in relation to pVT inducibility and maintenance. METHODS Sotalol was infused regionally or globally in isolated Langendorff blood-perfused pig hearts (N = 7) to create repolarization time (RT) heterogeneities. Programmed stimulation and epicardial activation and repolarization mapping were performed. The role of RT (heterogeneities) was studied in more detail using a computer model of the human heart. RESULTS pVTs (n = 11) were inducible at a critical combination of RT and RT heterogeneities. The pVT cycle lengths were similar in the short and long RT regions. Short-lasting pVTs were maintained by focal activity while longer-lasting pVTs by reentry wandering along the interface between the 2 regions. Local restitution curves from the long and short RT regions crossed. This was associated with T wave inversion at coupling intervals at either side of the crossing point. These experimental observations were confirmed by the computer simulations. CONCLUSION pVTs are inducible within a critical range of RT and RT heterogeneities and are maintained by reentry wandering along the repolarization gradient. Double potentials localize at the core of the reentrant circuit and reflect phase singularities. RT gradient and T waves invert with short-coupled premature beats in the long RT region as a result of the crossing of the restitution curves allowing reentry initiation

HCMV tegument protein pp71 and HPV minor capsid protein L2 can substitute transcriptional activation of the Ad genome.

<p>(A) H1299 cells were transfected with control vector, VI-wt, VI-M1 or VI-delta54 expression vector and subsequently infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Viral particles were harvested 24, 48 and 72 h p.i. and virus yield was determined using quantitative E2A stain. (B) Experimental setup as in A including the use of the same empty vector control except that cells were transfected with expression vector for the HCMV tegument protein pp71 or the HPV small capsid protein L2. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002549#s2" target="_blank">Results</a> are from three independent experiments. (C) U2OS cells were transfected with control vectors or expression vectors for VI-wt or VI-M1 as indicated in the legend together with control expression vectors for <i>Renilla</i> luciferase. Twenty four hours after transfection, cells were infected with MCMV encoding a firefly luciferase gene controlled by the HCMV immediate early promoter. Two hours after infection cells were lysed and firefly luciferase levels were measured and normalized for renilla luciferase expression by a dual luciferase assay. Expression levels were set to 100% for empty vectors. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002549#s2" target="_blank">Results</a> are the mean of two independent experiments performed in six technical repeats. Error bars represent the STD.</p

HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulate E1A promoter activation.

<p>(A) H1299 cells were transfected with luciferase reporter plasmids coding for E1A promoter and effector plasmids encoding for VI-wt, VI-M1, VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control. Forty eight hours after transfection, samples were lysed and luciferase activity was measured as described before. Mean and standard deviation are from three independent experiments. (B) H1299 cells were co-transfected with plasmids containing the Ad5 E1-region (pPG-S3) and expression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cell extracts were prepared 48 h after transfection and proteins were subjected to IB using Ab against RFP (pVI), pp71 or β-actin as indicated on the right. Note that several splice variants of E1A are recognized depicted by the vertical bar. (C) Cells were transfected as in B and indicated in the legend to C. Forty eight hours after transfection total RNA was prepared from cell lysates and reverse transcribed using oligo-dT primers. E1A mRNA levels were determined using qPCR with E1A specific, exon-spanning primers. Values correspond to the mean of two experiments done in triplicates and the error bar indicates the STD.</p